characterization of an abundant, unique 1.7-kilobase bovine

JOURNAL OF VIROLOGY,0022-538X/97/$04.0010

Jan. 1997, p. 527–538 Vol. 71, No. 1

Copyright q 1997, American Society for Microbiology

Characterization of an Abundant, Unique 1.7-Kilobase BovineHerpesvirus 4 (BHV-4) Late RNA and Mapping of a

BHV-4 IE2 Transactivator-Binding Site inIts Promoter-Regulatory Region†

ROSA BERMUDEZ-CRUZ,‡ LU ZHANG, AND VICKY L. VAN SANTEN*

Department of Pathobiology, Auburn University, Auburn, Alabama 36849-5519

Received 26 February 1996/Accepted 3 October 1996

We characterized an abundant late 1.7-kb cytoplasmic polyadenylated RNA (L1.7 RNA) transcribed from thebovine herpesvirus 4 (BHV-4) HindIII W fragment, in a region of the genome not conserved with Epstein-Barrvirus and herpesvirus saimiri. L1.7 RNA contains only extremely short (<100 nucleotides) open readingframes followed by two repeat arrays. The first repeat array contains 11 copies of a 23-bp unit, TGGCACTAGTAGCATTTAACCCC. The second and third copies are each interrupted by 15- to 17-bp sequences that areidentical to each other for the first 15 bp. In addition, the second and third copies of the repeat unit eachcontain two copies of nucleotides 5 to 9 (ACTAG) of the repeat unit, one at each end of the interruption. Thesecond repeat array contains 12 copies of a 25-bp sequence, GCTGTGTATTATTGAGTATTTTTTA. Thepromoter-regulatory region of L1.7 was activated by the BHV-4 immediate-early gene 2 product (IE2), ahomolog of the Epstein-Barr virus R transactivator, in cotransfection assays. We mapped an IE2 recognitionsite within a 167-bp fragment approximately 10 bp 5* to the start of L1.7 RNA transcription, using cotrans-fection assays and gel retardation assays. Using gel retardation assays, we mapped an IE2-binding site withinthis fragment to a 31-bp region from 56 to 86 bp 5* to the start of L1.7 RNA transcription. This IE2-bindingsite was able to transfer IE2 responsiveness to a heterologous promoter. However, IE2 responsiveness wasaffected by both position and orientation. Alignment of the L1.7 IE2-binding site sequence with sequences oftwo other BHV-4 IE2-binding sites resulted in a provisional IE2-binding site consensus sequence different fromthe Epstein-Barr virus R transactivator-binding site.

The genome of bovine herpesvirus 4 (BHV-4) shows homol-ogy to those of gammaherpesviruses Epstein-Barr virus (EBV)and herpesvirus saimiri (3). The genomes of these three virusesshare blocks of homologous genes; between the blocks of ho-mologous genes lie regions containing genes unique to eachvirus (3, 22). It has been suggested that genes unique to eachvirus determine the unique biological properties of each virus(3, 22).Like for other herpesviruses, the expression of BHV-4 genes

in infected cells is regulated. BHV-4 RNAs can be divided intoimmediate-early (IE), early (E), and late (L) classes accordingto their time of expression and sensitivity to inhibitors of pro-tein or viral DNA synthesis (6). Herpesvirus IE gene productsactivate expression of E and L genes. Two BHV-4 IE RNAshave been identified by characterization of viral RNAs in cellsinfected in the presence of a protein synthesis inhibitor (6, 34,35) (Fig. 1). IE RNA 1 is transcribed from a region of theBHV-4 genome not conserved among gammaherpesviruses,but it shows limited amino acid sequence homology to an IEprotein of the alphaherpesvirus herpes simplex virus type 1

(HSV-1), IE110, a nonspecific transactivator. However, no rolefor the BHV-4 IE1 gene product has been demonstrated bycotransfection assays (1, 35, 38). IE RNA 2 encodes a proteinwith amino acid sequence homology to the EBV R transacti-vator (35). The EBV R transactivator activates expressionfrom several EBV E promoter-regulatory regions via R-re-sponsive enhancers (7–9, 14–18, 23). The BHV-4 IE2 geneproduct is also a transactivator and activates expression fromseveral BHV-4 E promoters (1, 33, 35, 38). However, theBHV-4 IE2 transactivator does not activate an EBV target,and the EBV R transactivator does not recognize a BHV-4 IE2target (35). Therefore, although the EBV R transactivator andBHV-4 IE2 gene product exhibit amino acid sequence homol-ogy between their DNA-binding domains (35), their recogni-tion sites are different.To determine whether IE2 transactivates BHV-4 L genes in

addition to BHV-4 E genes, we have characterized an abun-dant 1.7-kb (L1.7) RNA and studied regulation of expres-sion from its promoter-regulatory region in cotransfectionassays. We chose the 1.7-kb RNA because of its abundanceand because it is transcribed from a region of the BHV-4genome not conserved among gammaherpesviruses. Wefound that the L1.7 RNA contains two tandem repeat arraysof two different, apparently non-protein-coding sequences.The promoter is transactivated by IE2 but very little, if at all,by IE1. Precise mapping of the IE2-binding site and com-parison to other IE2-binding sites enabled us to define aprovisional IE2-binding site consensus sequence. The IE2-binding site is able to transfer IE2 responsiveness to a het-erologous promoter, but responsiveness is affected by bothposition and orientation.

* Corresponding author. Mailing address: Department of Pathobi-ology, 264 Greene Hall, Auburn University, Auburn, AL 36849-5519.Phone: (334) 844-2668. Fax: (334) 844-2652. E-mail: [email protected].† Publication 2530 from the College of Veterinary Medicine, Au-

burn University.‡ Present address: Department of Genetics and Molecular Biology,

Centro de Investigacion y de Estudios Avanzados del IPN, Zacatenco,Mexico D.F. 07360.

527

MATERIALS AND METHODSCell culture, virus propagation, preparation of RNA, Northern (RNA) blot

analysis, S1 nuclease analysis, and primer extension analysis. The DN-599isolate of BHV-4 was used for all experiments unless otherwise noted. Isolate4N2 was isolated by cocultivation with BT (bovine turbinate) cells (ATCCCRL1390) of peripheral mononuclear leukocytes from a healthy BHV-4-sero-positive cow from the Auburn University College of Veterinary Medicine dairyherd. BT cells were used for transfection experiments and preparation of BHV-4-infected cell RNA. BT cells were cultured in Dulbecco’s modified Eagle me-dium containing penicillin, streptomycin, and 10% defined, supplemented bovineserum. Virus propagation, preparation of cytoplasmic polyadenylated RNA,Northern blot analysis, S1 nuclease analysis, and primer extension analysis wereperformed as previously described (34). Hybridization temperatures for S1 nu-clease analysis and primer extension analysis are noted in figure legends. Thehybridization temperature for S1 nuclease analysis using a double-strandedprobe with only 25% G1C content (129-bp SpeI-BfmI fragment) for whichresults are not shown was 308C; S1 nuclease treatment of samples containing thisprobe was at 158C for 1 h 15 min. S1 nuclease treatment of all other samples wascarried out at 228C for 45 min.DNA sequence analysis. Nucleotide sequence was determined by using a

Sequenase version 2.0 DNA sequencing kit (United States Biochemical Corpo-ration, Cleveland, Ohio) and double-stranded or single-stranded phagemid tem-plates. The sequence of the first repeat unit was confirmed by sequencing a PCRproduct of uncloned viral DNA template. Viral DNA was purified as previouslydescribed (34). PCR was carried out in 50-ml volumes containing approximately1 ng of denatured viral DNA, 25 pmol of each primer (identified in Fig. 3A), 1.5mM MgCl2, and other components of a GenAmp PCR Core Reagents kit(Perkin-Elmer Cetus, Norwalk, Conn.) according to instructions supplied withthe kit. PCR was carried out for 25 cycles of 1 min at 948C, 1 min at 518C, and1.5 min at 728C. Amplified DNA was isolated by electrophoresis on a 5%nondenaturing polyacrylamide gel, and 20 to 25% of each sample was sequencedby using each of the PCR primers and the fmole DNA sequencing system(Promega Corp., Madison, Wis.). Sequencing reactions were performed follow-ing instructions supplied with the kit, using 30 cycles of 30 s at 958C, 30 s at 498C,and 1 min at 708C.Transient expression cotransfection assays. Effector plasmids containing the

BHV-4 IE2 gene, pH12PR3.4, and the BHV-4 IE1 gene, pRSVIE1, used intransient expression cotransfection assays have previously been described (35).The control effector plasmid used was pTZ18U (United States Biochemical).Target plasmid pXH1.1CAT contains a 1.1-kb XmnI-HindIII fragment (Fig.

1A) inserted 59 to the promoterless chloramphenicol acetyltransferase (CAT)reporter gene in the pCAT-Basic vector (Promega). Target plasmidspDH844CAT, pNH507CAT, pKH301CAT, and pXS167CAT contain subfrag-ments of the 1.1-kb XmnI-HindIII fragment cloned into the pCAT-Basic vector.The letters in each plasmid name stand for restriction sites that define the endsof each fragment (see Fig. 4A), and the numbers indicate the number of nucle-

otides in the BHV-4 DNA fragment contained in each construct. The constructsalso contain additional nucleotides derived from the multiple cloning region ofthe pTZ19U vector. pXS56-120CAT contains a PCR-generated fragment includ-ing nucleotides (nt) 56 to 120 of the 167-bp XbaI-Sau3AI fragment (see Fig. 8E)cloned into pCAT-Basic.Target plasmids pCAT-Promoter and pCAT-Control were from Promega.

Target plasmids p31BF-CATP, p31NF-CATP, p31NR-CATP, p31BF-CATC,p31NF-CATC, and p31NR-CATC each contain a 31-bp fragment including nt 90to 120 of the 167-bp XbaI-Sau3AI fragment, generated by extending 39-comple-mentary synthetic oligonucleotides by using Klenow polymerase. The last letterin these plasmids names indicates whether the vector was pCAT-Promoter (P) orpCAT-Control (C). The letter B or N after the number indicates whether the31-bp fragment was inserted into the BglII site approximately 140 bp 59 to thesimian virus 40 (SV40) early major transcriptional start sites or into the NcoI siteapproximately 50 bp 59 to the major transcriptional start sites. The letter F or Rindicates whether the 31-bp fragment was inserted in forward or reverse orien-tation relative to the SV40 promoter.Plasmid DNA used for transfection was purified by alkaline lysis and polyeth-

ylene glycol precipitation (13), followed by CsCl-ethidium bromide equilibriumdensity gradient centrifugation (31). Residual RNA was removed by digestionwith RNase A and centrifugation of plasmid DNA through 1 M NaCl (31).Plasmid DNA was transfected into BT cells by calcium phosphate coprecipi-

tation as previously described (35), with the following exceptions: the calciumphosphate-DNA coprecipitate was incubated with cells for 18 to 20 h, after whichcells were rinsed with Dulbecco’s balanced salt solution, and medium waschanged once instead of twice. For experiments shown in Table 1, each trans-fection mixture (0.3 ml) contained 2.5 mg of target plasmid, 2.5 mg of effectorplasmid, and 2.5 mg of transfection efficiency control plasmid (pSV-b-Galacto-sidase; Promega). Infection of transfected cells with BHV-4 was carried out aspreviously described (35). For experiments shown in Fig. 4 and 9, each transfec-tion mixture (0.3 ml) contained 3.5 mg of target plasmid, 1.5 mg of effectorplasmid, and 2.5 mg of transfection efficiency control plasmid (pSV-b-Galacto-sidase). CAT assays were performed by the phase extraction procedure as pre-viously described (1, 32). b-Galactosidase assays for experiments shown in Table1 were performed with a colorimetric substrate as previously described (31, 35).b-Galactosidase assays for experiments shown in Fig. 4 and 9 were performedwith a chemiluminescent substrate as previously described (1, 20). To correct forrelative transfection efficiency, general level of gene expression in transfectedcells, and sample recovery, results are expressed as a ratio of CAT activity tob-galactosidase activity in arbitrary units, obtained by dividing the net counts perminute in the organic phase of each sample by the net luminometer reading foreach sample. In addition, results are also expressed uncorrected for b-galacto-sidase activity, simply as percent chloramphenicol acetylated.Transfections for evaluation of the 59 ends of RNA produced from target

plasmids were performed by calcium phosphate coprecipitation as describedabove except that cells were plated into 100-mm-diameter plates. Each transfec-tion mixture (1 ml) contained 12 mg of target plasmid, 5 mg of effector plasmid,and 8 mg of pTZ18U, added to bring the DNA concentration to 25 mg/ml.Approximately 48 h posttransfection, total RNA was harvested from the cells byusing 4.5 ml of RNA STAT-60 reagent (Tel-Test “B”, Inc., Friendswood, Tex.)/plate according to instructions supplied with the reagent. After purification,RNA was dissolved in 0.5% sodium dodecyl sulfate, heated at 688C for 20 min,and extracted with phenol-chloroform and chloroform prior to use in S1 nucleaseanalysis.Gel retardation assay. IE2 protein was produced in vitro either by separate in

vitro transcription and translation reactions (Fig. 5) or by coupled in vitrotranscription-translation reactions (Fig. 7 and 8) as previously described (38).32P-end-labeled DNA fragments (2 3 104 cpm) were incubated with 2 ml of invitro translation extract in a total volume of 15 ml for 30 min at room temper-ature as previously described (15) except that poly(dI-dC) was added to a finalconcentration of 0.13 mg/ml. Electrophoresis to separate IE2-DNA complexesfrom free DNA was performed also as previously described (15).Exonuclease III digestion of DNA for gel retardation assays. We labeled the

167-bp XbaI-Sau3AI fragment at either end after cutting plasmid containing thefragment with either XbaI or AvaI (in vector sequences) by adding [a-32P]dCTPto the 39 end with Klenow polymerase. The cut and labeled end was blunted byKlenow polymerase in the presence of 40 mM a-phosphorothioate-deoxynucleo-side triphosphate to block digestion by exonuclease III. The 167-bp fragment wasremoved from the vector by cutting with a second enzyme and isolated byelectrophoresis. The fragment was treated with 300 U of exonuclease III in 30 mlof 66 mM Tris-HCl (pH 8.0)–0.66 mMMgCl2 at room temperature. Samples (10ml) were removed after 2, 3, and 4 min and added to 30 ml of ice-cold buffercontaining 0.34 M NaCl, 0.04 M potassium acetate (pH 4.6), 1.35 mM zincacetate, and 300 U of S1 nuclease per ml. Samples were moved to room tem-perature for 30 min to allow S1 nuclease to digest the single-stranded portion ofthe fragment remaining after exonuclease III digestion. The reaction wasstopped by addition of 1/10 volume 0.3 M Tris base–0.05 M EDTA, and enzymeswere inactivated by heating at 708C for 10 min. Samples containing DNA treatedwith exonuclease III for different times were pooled and fractionated by elec-trophoresis in a 5% nondenaturing polyacrylamide gel. Size fractions were elutedfrom the gel and used for gel retardation assays. The size range of fragments in

FIG. 1. (A) Map positions of genes studied. A HindIII restriction map of theunique central portion of the BHV-4 (DN-599) genome is shown. The beginningof terminal tandem repeats is indicated by striped arrows. Above the map,expanded maps of the fragments encoding IE RNAs and L1.7 RNA are shown.The expanded map for IE RNA 1 and L1.7 RNA (L RNA 1.7) contains twoHindIII fragments, E and W, separated by a HindIII site (H). X, XmnI. (Onlyrelevant restriction sites are shown.) The black bar indicates the 1.1-kbXmnI-HindIII fragment in target plasmid pXH1.1CAT. All cloned DNA frag-ments used in this work were from the BHV-4 DN-599 isolate. (B) Restrictionfragments used as probes in Northern blot analysis and screening of a cDNAlibrary. A restriction map of HindIII-W (stippled portion) and part of HindIII-Eis shown. Abbreviations: S, SmaI; Ha, HaeIII; D, DraI; K, KpnI; H, HindIII. Thearrow represents the structure of L1.7 RNA, as determined by S1 nuclease andprimer extension analyses and by sequencing of cDNA. Bars below the map showrestriction fragments used as probes in Northern blot analysis. White bars indi-cate fragments that hybridized to both L1.7 RNA and the smaller RNA. Blackbars indicate fragments that hybridized only to L1.7 RNA. a, b, and c indicatefragments used as probes in screening the cDNA library (see text).

528 BERMUDEZ-CRUZ ET AL. J. VIROL.

each fraction was determined by electrophoresis on 6% polyacrylamide sequenc-ing gels.A 65-bp fragment comprising nt 56 to 120 of the 167-bp XbaI-Sau3AI frag-

ment cloned into the BamHI site of pTZ18U was labeled at either end andtreated with exonuclease III in the same way except that exonuclease treatmentwas for 1, 2, and 3 min. Fractions were isolated from an 8% polyacrylamide gel,and the size range of fragments in each fraction was determined by electrophore-sis on 8% polyacrylamide sequencing gels.Nucleotide sequence accession numbers. The L1.7 cDNA nucleotide sequence

has been submitted to GenBank under accession number U31371. The nucleo-tide sequence of the 1.1-kb XmnI-HindIII fragment containing the L1.7 RNApromoter-regulatory region corresponds to the complementary strand of nucle-otides 1102 to 1 of GenBank accession number M60043.

RESULTS AND DISCUSSION

Characterization of L1.7 RNA. The HindIII W fragment ofthe BHV-4 genome hybridizes to an extremely abundant1.7-kb RNA (L1.7 RNA) and a less abundant 1.2-kb RNA onNorthern blots of cytoplasmic polyadenylated RNA isolatedfrom BHV-4-infected cells late in infection (6). All subfrag-ments of HindIII-W tested hybridized to L1.7 RNA, but onlysubfragments from the left end of HindIII-W hybridized to the1.2-kb RNA (Fig. 1B). In addition, a 1.1-kb SmaI-HindIIIfragment to the left of HindIII-W hybridized to both RNAs(Fig. 1B). To identify a cDNA corresponding to L1.7 RNA, acDNA library (1) representing cytoplasmic polyadenylatedRNA of BHV-4-infected BT cells was initially screened byhybridization to two probes, the 1.1-kb SmaI-HindIII fragment(fragment a in Fig. 1B) and a 914-bp HaeIII fragment ofHindIII-W (fragment b in Fig. 1B). Two probes were used onduplicate filters to increase the likelihood of identifying near-full-length cDNAs. The library was made from RNA isolatedfrom infected cells 12 h postinfection (p.i.) and thus containsprimarily E viral RNA. However, viral DNA synthesis startsbefore 12 h p.i., and L1.7 RNA is already present at 12 h p.i.(6). One positive cDNA clone of many was characterized. Thenucleotide sequence of each end of the cDNA indicated thattranscription of the RNA begins to the left of HindIII-W, inHindIII-E, and continues through HindIII-W, and that theRNA contains sequences complementary to the first 15 nt ofHindIII I prior to the poly(A) tail (Fig. 1A). Because this1.4-kb cDNA is longer than the 1.2-kb RNA and containssequences corresponding to the right end of HindIII-W, whichdo not hybridize to the 1.2-kb RNA, this cDNA representsL1.7 RNA rather than the 1.2-kb RNA transcribed fromHindIII-W sequences. The size of restriction fragments of thecloned cDNA between the HindIII sites compared to HindIII-W genomic DNA was consistent with an unspliced RNA (datanot shown).The presence of a poly(A) tail in the cDNA not present in

the genomic DNA sequence (V. Test; EMBL nucleotide se-quence database accession number Z46380) beginning 14 nt 39of a polyadenylation signal (AATAAA) indicated that thecDNA was complete at the 39 end. We mapped the 59 end ofL1.7 RNA by S1 nuclease analysis and primer extension usingcytoplasmic polyadenylated RNA isolated 24 h p.i. The probefor S1 nuclease analysis and the primer for primer extensionwere 59-end labeled at the same site (Fig. 2B). Results indi-cated that transcription of L1.7 RNA begins approximately 100nt 59 to theHindIII site marking the left end ofHindIII-W. Thededuced sequence of a complete L1.7 cDNA is shown in Fig.3A.After mapping the 59 end of L1.7 RNA and characterizing its

promoter (see below), we screened the cDNA library againand characterized more cDNAs to see if they were similar tothe initial L1.7 cDNA characterized. To identify cDNAs cor-responding to RNAs transcribed from the L1.7 promoter, we

screened the cDNA library with a 301-bp KpnI-HindIII frag-ment probe (fragment c in Fig. 1B) which includes sequencescomplementary to approximately 100 nt at the 59 end of L1.7RNA. We characterized six positive clones by restriction en-zyme digestion and sequencing of the 39 end or both ends.Restriction enzyme digestion showed that four of the cDNAswere similar to the original L1.7 cDNA (not shown). There-fore, the initial cDNA characterized was representative of themajority of transcripts arising from the L1.7 promoter charac-terized below. However, in some cases there was minor sizeheterogeneity of fragments containing repeat arrays (Fig. 3A)among cDNAs. In addition, we observed minor size heteroge-

FIG. 2. (A) S1 nuclease and primer extension analysis of the 59 end of L1.7RNA. The hybridization temperature was 448C. Lanes: PE, primer extension; S1,S1 nuclease analysis; M, pBR322 DNA cleaved with MspI; U, uninfected cellRNA control; I, BHV-4-infected cell L RNA (prepared 24 h p.i.); P (second fromright), probe; P (right), primer. Marker sizes in nucleotides are indicated on theleft. Sizes in nucleotides of probe, primer, protected fragment, and primerextension product are indicated next to arrowheads on the right. (B) Schematicdiagrams of the double-stranded 59-end-labeled probe, protected fragment, dou-ble-stranded 59-end-labeled primer, primer extension product, and 1.7-kb RNAstructure. The right part of the restriction map shown corresponds to HindIII-W.The left part corresponds to 1.1 kb of HindIII-E.

VOL. 71, 1997 BHV-4 L1.7 RNA AND IE2-BINDING SITE 529

neity of fragments containing repeat arrays among subclonesof individual cDNAs, suggesting that the heterogeneity re-sulted from homologous recombination among repeat units inthe bacterial hosts. The sequences of the ends of three of thesefour cDNAs showed that the 39 ends were near the polyade-nylation site identified by sequence analysis of the initialcDNA, but each was apparently incomplete at the 39 end,missing 3 to 15 nt and lacking a poly(A) tail.The other two additional cDNAs characterized were consid-

erably shorter than the others but apparently do not representthe 1.2-kb RNA. The fifth additional cDNA is apparently anincomplete cDNA. It ends after nt 553 of the sequence shownin Fig. 3A. No polyadenylation signal is near this position, andthe cDNA lacks a poly(A) tail. Restriction digestion and par-tial sequencing of the sixth additional cDNA revealed a dele-tion of 489 bp relative to the original L1.7 cDNA, between nt668 to 671 and nt 1157 to 1160 (Fig. 3B). The exact endpointsof the deletion cannot be specified because the deletion ap-parently occurred between two short regions of identical se-quence. Overlapping the endpoints of the deletion, the originalcDNA sequence contains two 10-bp regions identical in ninepositions. Therefore, the deleted cDNA could have arisen

from a full-sized L1.7 cDNA via homologous recombinationmediated by these 10-bp regions in the bacterial host. Alter-natively, the deleted cDNA might represent an RNA tran-scribed from a deleted viral template which arose via homol-ogous recombination. Yet another possibility is that the“deleted” cDNA represents a spliced RNA. The 59 end of thedeletion is flanked by a sequence conforming to the 59 splicesite consensus sequence at five of eight positions, and the 39end of the deletion is flanked by a sequence conforming verywell to the 39 splice site consensus sequence (Fig. 3B) (27).Splicing utilizing these two potential splice sites would result inthe sequence found in the “deleted” cDNA. Therefore, weperformed S1 nuclease analysis using a 39-end-labeled SpeI-BfmI fragment probe extending from positions 564 to 694 ofthe sequence shown in Fig. 3A to determine whether the po-tential 59 splice site at position 671 is used. Cytoplasmic poly-adenylated RNA from BHV-4-infected cells protected onlyfull-length probe. No protected probe fragment indicatingRNA spliced at position 671 was detected (data not shown).Therefore the “deleted” cDNA most likely arose by a homol-ogous recombination event. In addition, the results of the S1

FIG. 3. (A) Deduced nucleotide sequence of complete L1.7 cDNA. The sequence was determined by using a Sequenase version 2.0 DNA sequencing kit anddouble-stranded or single-stranded templates derived from cDNA and genomic DNA plasmid clones. Nucleotides 1 to 8 and 552 to 977 were determined from genomicclones only; nt 9 to 551 and 978 to 1333 were determined from genomic and cDNA clones (genomic and cDNA sequences were identical); nt 1334 to 1356 weredetermined from cloned cDNA only. S1 nuclease analysis (Fig. 2 and data not shown) and size of restriction fragments of cDNAs indicated that there were no splicesin the portions of the sequence determined from genomic clones only. An array of 23-bp repeats is underlined; interruptions are indicated by italics; the beginning ofeach repeat unit is indicated by a dot over the sequence. An array of 25-bp repeats is double underlined; the beginning of each repeat unit is indicated by a diamondover the sequence. The putative polyadenylation signal is indicated by asterisks. Predicted amino acid sequences of two short ORFs are indicated below the nucleotidesequence. Differences in the V. Test isolate DNA sequence (EMBL nucleotide sequence database accession number Z46380) are shown as lowercase letters above thenucleotide sequence. The triangle indicates a 27-bp insertion in V. Test compared to DN-599. The dashes indicate deletions in V. Test compared to DN-599. In addition,the V. Test isolate sequence has more copies of the 23-bp repeat than DN-599. However, it contains the same number of 25-bp repeats as DN-599. Location of primersused for PCR of uncloned genomic DNA and sequencing of PCR product to verify the sequence of the 23-bp repeat are indicated by a line over the sequence. (B)Sequence across the endpoints of the deletion of the “deleted” cDNA. The L1.7 cDNA 13 (referred to as the sixth additional cDNA and the deleted cDNA in the text)sequence is aligned with portions of the L1.7 cDNA sequence shown in panel A, L1.7 cDNA1. Numbers on the right show position numbers in the sequence in panelA. Vertical lines indicate identity. Asterisks above and below the L1.7cDNA1 sequence indicate identical nucleotides which could have mediated deletion byhomologous recombination. Underlining indicates nucleotides matching splice site consensus sequences. Boxes indicate exons if the potential splice sites were used.


nuclease analysis showed that the polyadenylation signal (AATAAA) at positions 627 to 632 is not used at detectable levels.The sequence of DNA encoding L1.7 RNA (Fig. 3A) does

not contain long open reading frames (ORFs). Two shortORFs, each less than 40 codons, are present in the 59 portionof the RNA. The 59-most ORF is the 39 portion of a 63-codonORF beginning 74 nt 59 to the start of transcription of L1.7RNA. The center portion of L1.7 RNA is occupied by tworepeat arrays. The first repeat array contains 11 copies of a23-bp unit, TGGCACTAGTAGCATTTAACCCC. The sec-ond and third copies are each interrupted by 15- to 17-bpsequences that are identical to each other for the first 15 bp. Inaddition, the second and third copies of the repeat unit eachcontain two copies of nt 5 to 9 (ACTAG) of the repeat unit,one at each end of the interruption. One of the short ORFsterminates in the second copy of the repeat. The repeatedsequence contains a stop codon in two of the three readingframes. Because the length of the repeat is not a multiple ofthree nucleotides, the ORF cannot be maintained through therepeat array. The second repeat array is separated from thefirst repeat array by 143 nt. The second repeat array contains12 copies of a 25-bp sequence, GCTGTGTATTATTGAGTATTTTTTA. Because the length of the repeat is not a multipleof three nucleotides and the repeated sequence contains a stopcodon in one reading frame, the second repeat array is alsoapparently non-protein coding.The presence of an abundant polyadenylated RNA that ap-

parently has little protein-coding potential in the cytoplasm ofinfected cells is puzzling. To confirm that the number of basesin the first repeating unit is not a multiple of 3, we sequencedeach strand of the first repeat array by using 7-deaza-dGTPinstead of dGTP to resolve possible band compression arti-facts. Two templates were sequenced. Both were PCR prod-ucts derived from uncloned viral DNA, in case the number ofnucleotides in the repeat sequence was a cloning artifact. OnePCR product was derived from DN-599 DNA; the other wasderived from a fresh isolate of BHV-4, 4N2, in case the numberof nucleotides in the repeat unit had changed over passage inculture. The sequences of the repeat units of both DN-599 and4N2 viral DNA were identical to the sequence of the repeatunit of cloned DN-599 viral DNA, revealing no sequencing,cloning, or passage artifacts. The sequence of the repeatingunit is the same for DN-599, 4N2, and V. Test isolates, but thenumber of repeating units varies. Isolate 4N2 has at least 21uninterrupted copies of the 23-bp repeat, V. Test has 13, andDN-599 has only 8. Others have already noted the heteroge-neity in size of this region of the genome among BHV-4 iso-lates (4). Although use of 7-deaza-dGTP did not reveal anyerrors in determining the number of nucleotides in the repeatunit, one observation is not inconsistent with continuation ofan ORF through the first repeat array. Immediately followingthe 23-bp repeats, our DN-599 sequence has a 27-bp deletioncompared to the V. Test sequence. If this were part of an ORF,the reading frame would be maintained by deletion or inser-tion of this multiple of three nucleotides.Activation of the L1.7 RNA promoter-regulatory region by

the BHV-4 IE2 gene product. To determine whether BHV-4 IEgene products activate expression of L1.7 RNA, we con-structed a reporter gene plasmid (pXH1.1CAT) by inserting a1.1-kb XmnI-HindIII fragment (Fig. 1A) 59 to the promoterlessCAT gene in pCAT-Basic vector (Promega). This fragmentbegins 1 kb 59 to the start of transcription of L1.7 RNA andcontains approximately 100 bp of DNA encoding the 59 portionof L1.7 RNA. In transient expression cotransfection assays,BHV-4 infection increased CAT expression from pXH1.1CATover 100-fold (Table 1). To determine whether the BHV-4 IE1

or IE2 gene product activates expression from the L1.7 pro-moter-regulatory region, we transfected cells with target plas-mid pXH1.1CAT, an effector plasmid encoding IE1 or IE2, ora vector control, plus a transfection efficiency control plasmidencoding b-galactosidase. Cotransfection with a plasmid en-coding IE2 stimulated CAT expression nearly 300-fold com-pared to the vector control relative to b-galactosidase expres-sion from the transfection efficiency control plasmid (Table 1).Cotransfection of a plasmid encoding IE1 stimulated CATexpression only fourfold, and cotransfection of IE1 and IE2plasmids apparently did not produce a synergistic effect (Table1). However, cotransfection of an IE1 plasmid decreased theamount of b-galactosidase expressed from the transfection ef-ficiency control plasmid (Table 1), making interpretation of theresults difficult.Therefore, we have demonstrated that the BHV-4 IE2 trans-

activator stimulates expression from a BHV-4 L promoter inaddition to BHV-4 E promoters. Whether the EBV R trans-activator is involved in activation of EBV L genes has not beentested. However, HSV-1 IE175, an essential specific transacti-vator, activates both E and L HSV-1 promoters (2, 10, 11, 19,24–26, 28, 29) and is continuously required to maintain L geneexpression (36). Our results show that transactivation of Lgenes by IE gene products also occurs in the gammaherpesvi-rus subfamily.Mapping of the IE2 response element(s). To map the IE2

response element(s) within the 1.1-kb XmnI-HindIII fragment,we carried out cotransfection assays using target plasmids con-taining subfragments of the 1.1-kb fragment cloned into thevector containing the promoterless CAT gene. Subfragmentsand results are shown in Fig. 4. IE2 stimulated expression ofCAT activity from reporter constructs 1 to 4, indicating that anIE2 response element is present in the 301-bp KpnI-HindIIIfragment. Constructs 5 and 6 were designed based on results ofgel retardation assays and will be discussed below.Mapping of the IE2-binding site.We have previously shown

that IE2 is a sequence-specific DNA-binding protein whichforms complexes with DNA fragments containing the BHV-4thymidine kinase (TK) gene IE2 response element in gel re-tardation assays (38). We used gel retardation assays to mapthe IE2-binding site within the 301-bp KpnI-HindIII fragmentcontaining the L1.7 IE2 response element identified by co-transfection assays. The results (Fig. 5) showed that the IE2-binding site is within a 167-bp XbaI-Sau3AI fragment fromapproximately 2177 to 27 relative to the start of L1.7 RNAtranscription. Failure of the 106-bp fragment to form an IE2-specific complex confirms that the complex formation is DNAsequence specific. The 167-bp XbaI-Sau3AI fragment was suf-

TABLE 1. Transactivation of target plasmid pXH1.1CAT byBHV-4 infection and cotransfection of BHV-4

IE1 or IE2 expression plasmidsa

Effector% Chloram-phenicolacetylated

RelativeCATactivity

b-Galactosi-dase activity(106 U/ml)

RelativeCAT activitycorrected forb-galactosi-dase activity

Vector control 0.11 1.0 4.74 1.0BHV-4 infection 39.32 344.0 12.19 133.7IE1 0.05 0.5 0.53 4.1IE2 23.12 202.3 3.23 296.9IE1 1 IE2b 7.15 62.6 0.82 362.9

a Values shown are averages for duplicate samples.b Half of the amount of each effector plasmid was present as when each

effector plasmid was used alone.


ficient to mediate activation by IE2 in cotransfection assayswhen linked to a promoterless CAT gene (Fig. 4, line 5) andtherefore contains an IE2-responsive promoter. This frag-ment contains the sequence TATAAAA, flanked by GC-richsequences, 24 to 30 bp 59 to the start of L1.7 RNA tran-scription, which likely functions as a TATA box for L1.7 RNA.S1 nuclease analysis of RNA from cells transfected withpXS167CAT target plasmid and IE2 effector plasmid (Fig. 6,lanes 1 to 4) showed that the increase in CAT activity in thepresence of IE2 was due to increased levels of RNA with 59ends in vector sequences approximately the same distance 39 tothe L1.7 TATA box as the 59 ends of L1.7 RNA transcribedfrom the intact BHV-4 genome in BHV-4-infected cells. Probefragment protected by this RNA transcribed in the absence ofIE2 was barely detectable and cannot be seen in the reproduc-tion of the autoradiograph shown in Fig. 6 (lane 3). However,probe fragment protected by this RNA transcribed in the pres-ence of IE2 was readily detected (Fig. 6, lane 4).We mapped the IE2-binding site within the 167-bp fragment

by digesting the fragment from either end with exonuclease IIIand S1 nuclease and testing size fractions for IE2 binding bygel retardation assay. The results (not shown) identified a65-bp fragment, nt 56 to 120 of the 167-bp fragment, contain-ing sequences necessary for IE2 binding. Gel retardation as-

says using the 65-bp fragment confirmed that it contains se-quences sufficient for IE2 binding (Fig. 7). However, thisfragment does not contain the putative L1.7 TATA box, andwhen inserted 59 to a promoterless CAT gene, the 65-bp frag-ment did not lead to activation of CAT expression in thepresence of IE2 (Fig. 4, line 6). This finding suggests that thefragment does not contain promoter sequences necessary forefficient transcription.A complex with an endogenous protein(s) in the rabbit re-

ticulocyte lysate used for in vitro translation of IE2 proteininterfered with detection of IE2-specific complexes withsmaller DNA fragments and prevented mapping the IE2-bind-ing site more precisely than within the 65-bp fragment, usingexonuclease III-generated size fractions in gel retardation as-says with IE2 protein generated by in vitro transcription fol-lowed by in vitro translation. However, production of IE2 bycoupled in vitro transcription-translation resulted in higherlevels of IE2, leading to more IE2-specific complex relative tothe complex with endogenous protein in gel retardation assays.This enabled us to detect IE2-specific complexes with smallerDNA fragments, and we undertook another series of exonu-clease III digestion-gel retardation experiments starting withthe 65-bp fragment identified by the first series of experiments.Most of the fractions of DNA digested from the left end still

FIG. 4. Mapping of the L1.7 RNA IE2 response element(s) by transient expression cotransfection assays. (A) A restriction map of the region 59 to L1.7 RNA isshown at the top. Abbreviations: Xm, XmnI; D, DraI; N, NcoI; K, KpnI; Xb, XbaI; S, Sau3AI; H, HindIII. Restriction sites shown are not all unique but are the relevantones. The nucleotide sequence of this entire fragment is known (34). The transcriptional start site that was identified by S1 nuclease and primer extension analysis (Fig.2) is indicated by the small arrow. Below the restriction map, the structures of target plasmids used in cotransfection assays are shown. Black bars indicate fragments59 to the L1.7 gene that were cloned 59 to a CAT reporter gene (striped bars) in pCAT-Basic vector. To the right of the target plasmid diagrams, a summary of resultsof cotransfection assays for each target plasmid is shown. Gray bars indicate CAT activity from the target plasmid in the absence of an IE2 effector plasmid relativeto b-galactosidase activity from a control plasmid. Striped bars indicate CAT activity from the target plasmid in the presence of an IE2 effector plasmid relative tob-galactosidase activity from the control plasmid. Results are the averages of six samples (three independent experiments with duplicate samples in each experiment)6 standard error. To the right of the graph, fold activation by IE2 for each target plasmid is indicated. Values were calculated by the following formula: average (CATactivity in the presence of IE2/b-galactosidase activity in the presence of IE2)/average (CAT activity in the absence of IE2/b-galactosidase activity in the absence ofIE2). (B) Results of experiments shown in panel A but without correction based on b-galactosidase activity. Fold activation values were calculated as average percentchloramphenicol converted in samples with IE2/average percent chloramphenicol converted in samples without IE2. Fold activation values based on corrected valuesare higher than those based on uncorrected values because b-galactosidase activities are lower in the presence of IE2, presumably due to squelching.


formed complexes with IE2 (Fig. 8A, fractions 1A to 8A),suggesting that most of the sequences at the left end of thefragment were not necessary for IE2 binding. However, frac-tions lacking DNA to the right of position 90 (fractions 9A and10A) did not form complexes with IE2, suggesting that se-quences to the right of position 90 were essential for IE2complex formation. In contrast, most of the fractions of DNAdigested from the right end formed complexes with IE2 poorly(Fig. 8C, fractions 4X to 6X) or not at all (fractions 7X to10X). Fractions that bound IE2 efficiently (fractions 1X to 3X)had only vector and primer sequences removed and containedall of the BHV-4 sequences (Fig. 8E). Combined, results ofIE2 binding of fractions of DNA digested with exonuclease IIIfrom either end suggested that the 31-bp segment including nt90 to 120 of the 167-bp XbaI-Sau3AI fragment contained se-quences necessary for IE2 binding (Fig. 8E). Removal of por-tions of the 31-bp segment from either end abolished or se-verely reduced IE2 binding (fractions 9A, 10A, and 4X to10X). Gel retardation assays using the 31-bp fragment (Fig. 7)confirmed that it contains sequences sufficient for IE2 binding.

FIG. 5. Mapping of the L1.7 IE2 recognition site(s) by IE2 complex forma-tion in vitro. (A) Gel retardation assays. DNA fragments used are indicated bytheir sizes in base pairs above each panel. Map positions of each fragment areindicated in panel B. Lanes 1, 4, 7, and 10 contain no in vitro translation extract.Lanes 2, 5, 8, and 11 contain in vitro translation extract without added RNA.Lanes 3, 6, 9, and 12 contain in vitro translation extract with in vitro-transcribedIE2 RNA. IE2-specific complexes (formed only in the presence of IE2) areindicated by arrowheads. NS indicates non-IE2-specific complexes, formed alsoin the absence of IE2. F indicates free DNA fragments. DNA fragments were notlabeled to the same specific activity, and exposures shown represent differentlengths of time. (B) Map positions of fragments used in gel retardation assays. Arestriction map of the region surrounding the start of transcription of L1.7 RNAis shown. Abbreviations are the same as in Fig. 4 except that X represents XbaI.The start of transcription of L1.7 RNA is indicated by the small arrow. The ORFis not drawn to scale. Fragments used in gel retardation assay are indicated aswhite and black bars below the map. The size of each fragment is indicated inbase pairs. Black bars indicate fragments that form complexes with IE2, andwhite bars indicate fragments that do not. The IE2 complex formed with the273-bp fragment is not as clearly visible as the IE2 complex formed with the167-bp fragment. However, because the 167-bp fragment is a subfragment of the273-bp fragment, the 273-bp fragment must contain an IE2-binding site(s). Weoften observe clearer IE2 complexes with smaller DNA fragments (unpublishedresults). Perhaps larger fragments contain more binding sites for additionalproteins present in the rabbit reticulocyte lysate used for in vitro translation, andadditional complexes obscure results.

FIG. 6. (A) S1 nuclease analysis of 59 ends of RNA transcribed from targetplasmids activated by IE2 in cotransfection assays. Target plasmids are indicatedat the top. Lanes: P, probe; M, pBR322 DNA cleaved withMspI; N, no RNA; 2,total RNA (25 mg) from cells transfected with target plasmid and pTZ18U vectorcontrol;1, total RNA (25 mg) from cells transfected with target plasmid and IE2plasmid. Probes were generated by PCR using one 59-end-labeled primer. Hybrid-ization temperatures were 448C for lanes 2 to 4 and 468C for lanes 6 to 8 and 10 to12. The autoradiograph of the gel was scanned by a Hewlett-Packard ScanJet IIcwith DeskScan II version 1.61 software. All lanes shown are from the same geland same autoradiograph, but more sensitive scans of lanes 6 to 8 and 10 to 12 areshown. Marker sizes in nucleotides are indicated in the center. Sizes in nucleo-tides of probes and protected fragments are indicated next to arrowheads at theright and left. In lanes 3 and 4, RNA transcribed from pXS167CAT initiated invector sequences at the same position relative to L1.7 promoter sequences asRNA transcribed from the viral genome in infected cells would protect a 78-ntprobe fragment. The origin of the extra 252-bp probe fragment present in lanes 9 to12 is unknown. However, its presence did not affect results, because identical pro-tected fragments were produced in experiments using probe preparations not con-taining the extra fragment (not shown). (B) Schematic diagrams of templates andprimers for generation of double-stranded 59-end-labeled probes by PCR, probes,and protected fragments. In the diagrams of templates, the stippled bars and blackbars represent BHV-4-derived sequences, the black bars represent the 31-bp frag-ment which contains an IE2-binding site demonstrated by gel retardation assay,vertically striped bars represent the vector fragment containing the enhancerlessSV40 early promoter, and the white bars represent other vector sequences. Thesmall arrows labeled E and L indicate the major start sites of transcription from theSV40 early promoter at early and late times of infection, respectively (12). Asterisksrepresent the labeled end of primers, probes, and protected fragments. Sizes innucleotides of probes and protected fragments are indicated on the right.


Effect of the IE2-binding site on a heterologous promoter.To determine whether the L1.7 IE2-binding site is sufficient totransfer IE2 responsiveness to a heterologous promoter, weinserted the 65- and 31-bp fragments, shown above to eachcontain an IE2-binding site, into the pCAT-Promoter vector ineither orientation approximately 140 bp 59 to the start of tran-scription of CAT RNA from the enhancerless SV40 early pro-moter. The fragments were also inserted into the pCAT-Con-trol vector, which contains the SV40 early promoter andenhancer. We tested these reporter gene constructs for activa-tion by IE2 in cotransfection assays. IE2 transactivated CATexpression very modestly (approximately threefold) from thesereporter gene constructs compared to transactivation of ex-pression from pXS167CAT, included in each experiment as apositive control (several hundred-fold; data not shown). Theseresults suggested that the L1.7 IE2-binding site was not suffi-cient to confer IE2 responsiveness to the SV40 early promoter.We have also observed failure of IE2-binding sites derived

from other BHV-4 promoters to transfer IE2 responsiveness tothe SV40 early promoter (1, 33). One possible explanation isthat, unlike EBV R-binding sites, the function of IE2-bindingsites in IE2 activation of gene expression is position depen-dent. This possibility is suggested by our observation that theIE2-binding sites of the L1.7 promoter and three other BHV-4promoters that have been mapped precisely all fall within 86 bpof the start of transcription (1, 33, 38). This is in contrast toEBV R transactivator-binding sites, which have been mappedas far as over 700 bp 59 to the start of transcription (15); mostEBV R-binding sites map over 300 bp 59 to the start of tran-scription (5, 9, 14, 21). Position dependence could explain thefailure of the L1.7 IE2-binding site to transfer IE2 responsive-ness to a heterologous promoter. The site of insertion in ourconstructs, the BglII site 140 bp 59 to start of transcription,might be too far away for efficient action of IE2.Therefore, to determine whether the L1.7 IE2-binding site

could transfer IE2 responsiveness to a heterologous promoterif inserted closer to the start of transcription, we inserted the31-bp fragment containing the L1.7 IE2-binding site into thepCAT-Promoter and pCAT-Control vectors at the NcoI siteapproximately 50 bp 59 to the start of transcription. In theforward orientation, this places the 31-bp fragment at positions254 to284 relative to the start of transcription, very similar toits position relative to the start of transcription in the intactL1.7 promoter (256 to 286). Results of cotransfection exper-iments using these reporter plasmids are presented in Fig. 9.Insertion of the IE2-binding site into the NcoI site of pCAT-Control decreased the promoter activity in the absence of IE2(Fig. 9; compare lines 8 and 9 with line 6). However, thepresence of IE2 restored promoter activity. The decrease ofpromoter activity in the absence of IE2 caused by the insertionof the 31-bp fragment could be due to disruption of promoterelements required for optimal expression. Alternatively, theIE2-binding site or other sequences in the fragment mightcontain recognition sites for inhibitory factors acting in theabsence of IE2. This would provide an additional mechanismof control of BHV-4 gene expression.Reporter constructs with the IE2-binding site inserted into

the NcoI site, between positions 254 and 284 relative to thestart of transcription, were more responsive to IE2 than thereporter constructs with the IE2-binding site inserted into theBglII site 140 bp 59 to the start of transcription. However, thefold activation mediated by the IE2-binding site in its naturalcontext, within the 167-bp fragment, was over 10 times greaterthan the fold activation of the most IE2-responsive reporterconstruct containing the 31-bp fragment and the SV40 earlypromoter (p31NF-CATC [Fig. 9, line 8]). In addition, the ac-

FIG. 7. Gel retardation assays using IE2-binding fragments identified byexonuclease III digestion. Fragment 65 contains 65 bp (uppercase letters in Fig.8E) from 120 to 56 bp 59 to the start of L1.7 RNA transcription generated byPCR, plus vector sequences. It is the same as fragment 0X in Fig. 7 and was 32Plabeled at one 39 end. Fragment 31 contains 31 bp (underlined uppercase lettersin Fig. 8E) from 86 to 56 bp 59 to the start of L1.7 RNA transcription, plusadditional nucleotides providing restriction sites for subsequent cleavage andcloning. It was generated by extending 39-complementary synthetic oligonucle-otides by using Klenow polymerase, dATP, dGTP, dTTP, and [a-32P]dCTP.Fragment 31 is therefore labeled to a higher specific activity than fragment 65.Lanes 1 and 4 contain no in vitro translation extract. Lanes 2 and 5 containcoupled in vitro transcription-translation reaction mixtures without added tem-plate. Lanes 3 and 6 contain coupled in vitro transcription-translation reactionmixtures containing IE2 cDNA template. Unbound (Free [F]) fragment contain-ing the 31-bp sequence ran off the gel. The autoradiograph was scanned by aHewlett-Packard ScanJet IIc with DeskScan II version 1.61 software.

FIG. 8. Mapping of the IE2-binding site by using size fractions of exonuclease III-treated DNA in gel retardation assays. (A) Plasmid DNA containing the 65 bpfrom bp 120 to 56 59 to the start of L1.7 RNA transcription was digested with AvaI in vector sequences and 39-end labeled, and the labeled end was blocked fromexonuclease III digestion by filling in with a-phosphorothioate-deoxynucleotide triphosphates. After cleavage with HindIII in vector sequences on the other side of thecloned fragment, DNA was treated with exonuclease III and S1 nuclease. Ten size fractions were isolated from a nondenaturing polyacrylamide gel and used in gelretardation assays. Lanes: 2, coupled in vitro transcription-translation reaction with no IE2 cDNA added; 1, coupled in vitro transcription-translation reactioncontaining IE2 produced from IE2 cDNA. Fractions are indicated above each set of lanes and shown schematically in panel E. Fraction 0A is fragment not treatedwith exonuclease III. Unbound fragments 8A, 9A, and 10A ran off the gel. The IE2-specific complex formed with DNA in fraction 0A is marked with an arrowheadat the left. IE2-specific complexes formed with smaller fractions migrated faster. IE2-specific complexes formed with fractions 6A, 7A, and 8A appear to migrate astwo bands, neither of which comigrates exactly with a dimmer band seen in lanes without IE2. (B) The size range of each fraction was determined by electrophoresison a denaturing polyacrylamide gel. M is pBR322 DNA cleaved with MspI. (C) Gel retardation analysis of exonuclease III/S1 nuclease-treated cloned 65-bp fragmentlabeled at the XbaI site in vector sequences. Lanes are the same as in panel A. Fraction 0X is fragment not treated with exonuclease III. Unbound DNA fragmentsran off the gel. The IE2-specific complex formed with DNA in fraction 0X is marked with an arrowhead at the left. IE2-specific complexes formed with smaller fractions(1X to 3X) migrated slightly faster. (D) The size range of each fraction was determined by electrophoresis on a denaturing polyacrylamide gel. (E) Schematic diagramof size fractions and summary of results. The double-stranded DNA sequence of the cloned 65-bp fragment (nt 56 to 120 of the 167-bp XbaI-Sau3AI fragment) is shownat the top. Vector- and primer-derived bases are indicated in lowercase letters. Fractions that form IE2-specific complexes efficiently are shown as dark bars; fractionsthat do not form IE2-specific complexes efficiently are shown as light bars. The heterogeneity in size of each fraction is indicated by the dotted portion of each bar.Results indicate that the underlined portion of the sequence contains sequences necessary for efficient IE2-specific complex formation. When these sequences wereremoved, IE2-specific complexes were not formed efficiently.


tivity of the IE2-binding site appeared to be affected by orien-tation. In both the pCAT-Promoter and pCAT-Control vec-tors, activation by IE2 was higher when the IE2-binding sitewas inserted in a forward orientation relative to the SV40promoter (compare lines 4 and 5 and lines 8 and 9). In thepresence of IE2, expression of CAT from the SV40 promoterwas highest when both the SV40 enhancer and the IE2-bindingsite were present (line 8). The dependence of the transactiva-tion activity of IE2 on the position and orientation of its rec-ognition site supports our suggestion that, unlike the EBV Rtransactivator, BHV-4 IE2 functions as a promoter factorrather than an enhancer factor (1).The IE2-binding site may mediate transactivation more ef-

fectively in its natural context than when linked to the SV40promoter because IE2 activation requires interaction withother transcription factors with recognition sites in BHV-4promoter-regulatory regions. We have been able to transfer

IE2 responsiveness to a heterologous promoter by insertion ofmultiple copies of an IE2 response element from the BHV-4TK promoter into the BglII site approximately 140 bp 59 to thestart of transcription in the pCAT-Promoter vector (38). In thiscase, multiple copies of an IE2-binding site might lead tobinding of multiple IE2 molecules, overcoming the need foradditional transcription factors or compensating for weak ac-tivity of IE2 at a greater distance from the start of transcrip-tion.To determine whether the increase in CAT activity from

p31NF-CATC observed in the presence of IE2 is mediated byan increase in the amount of RNA transcribed from a specifictranscription start site, we mapped the 59 ends of transcripts intransfected cells by S1 nuclease analysis (Fig. 6). RNA fromcells transfected with pCAT-Control was included for compar-ison. We found, in agreement with results of others (12), thatRNA transcribed from the SV40 early promoter has hetero-

FIG. 9. Effect of the L1.7-binding site on SV40 promoter activity in cotransfection assays. (A) Names and schematic diagrams of target plasmids are shown on theleft. pXS167CAT (see Fig. 4) was included in all experiments for comparison. In the diagram of pXS167CAT, the stippled bar and black bar represent BHV-4-derivedsequences. In all diagrams, the black bar and large black arrows represent the 31-bp fragment which contains an IE2-binding site demonstrated by gel retardation assay.The transcriptional start sites are indicated by the small arrows. Target plasmids 3 to 5 contain the 31-bp fragment inserted into pCAT-Promoter vector (line 2), andtarget plasmids 7 to 9 contain the 31-bp fragment inserted into the pCAT-Control vector (line 6). Orientation is indicated by the direction of the large black arrows.Abbreviations: B, BglII; N, NcoI. The bar with vertical stripes represents the vector fragment containing the enhancerless SV40 early promoter. The asterisks indiagrams 6 to 9 represents the SV40 enhancer, approximately 2 kb 59 to the SV40 promoter fragment. White portions of the diagrams represent other vector-derivedsequences. To the right of the target plasmid diagrams, a summary of results of cotransfection assays for each target plasmid is shown. Gray bars indicate CAT activityfrom the target plasmid in the absence of IE2 effector plasmid relative to b-galactosidase activity from a control plasmid. Striped bars indicate CAT activity from thetarget plasmid in the presence of IE2 effector plasmid relative to b-galactosidase activity from the control plasmid. Results are the averages of six samples (threeindependent experiments with duplicate samples in each experiment) 6 standard error. To the right of the graph, fold activation by IE2 for each target plasmid isindicated. Values were calculated by the following formula: average (CAT activity in the presence of IE2/b-galactosidase activity in the presence of IE2)/average (CATactivity in the absence of IE2/b-galactosidase activity in the absence of IE2). (B) Results of experiments shown in panel A but without correction based onb-galactosidase activity. Fold activation values were calculated by average percent chloramphenicol converted in samples with IE2/average percent chloramphenicolconverted in samples without IE2. Fold activation values based on corrected values are higher than those based on uncorrected values because b-galactosidase activitiesare lower in the presence of IE2, presumably due to squelching.


geneous 59 ends. RNA transcribed from pCAT-Control pro-tected fragments of at least three different sizes (fragments 3,4, and 5 in Fig. 6, lanes 7 and 8). Fragments 4 and 5 correspondto the sizes expected of fragments protected by RNAs initiatedat the SV40 early transcription start sites (12). In contrast tothe results shown in Fig. 9, line 6, which would predict adecrease in the amount of CAT RNA in the presence of IE2,in this experiment, the presence of IE2 increased the amountof CAT RNA specifically initiated from pCAT-Control. RNAtranscribed from p31NF-CATC in the presence of IE2 pro-tected the same probe fragments protected by RNA tran-scribed from pCAT-Control and two additional fragments(fragments 1 and 2 [Fig. 6, lane 12]). The additional fragmentsdo not correspond to sizes expected if protected by RNAinitiated at previously identified transcription start sites in theSV40 early promoter region, including RNAs transcribed inthe early direction at late times after infection (12). Therefore,insertion of an IE2 binding site into the NcoI site of the SV40promoter region affected the sites of initiation of transcriptionin the presence of IE2, in addition to increasing the amount ofRNA. Because RNA transcribed from p31NF-CATC in theabsence of IE2 was not detectable (Fig. 6, lane 11), we werenot able to determine whether insertion of the IE2 binding siteinto the SV40 promoter caused the change in transcriptioninitiation sites, and IE2 only increased the amount of transcrip-tion from these sites, or whether IE2 caused the change intranscription initiation sites.IE2-binding site consensus sequence. Mapping of several

IE2-binding sites precisely has allowed us to identify a provi-sional consensus sequence for IE2-binding sites, based on se-quence similarities among the IE2-binding fragments (Fig. 10).The EBV R-binding site consensus sequence, G(G/T)CCN7GTGGTG (16), consists of two parts separated by 7 bp. Such apattern is not evident in our consensus sequence. However, theEBV R-binding site consensus sequence was based on muchmore extensive work, comparison of over 45 R-binding sitesand methylation interference studies to determine which basescontact the R transactivator (14–16, 30). Our IE2-binding siteconsensus sequence does not address which bases contact theprotein. However, mutagenesis of the L1.7 IE-binding site hasshown that bases 2 to 5 of the third box of our consensussequence are required for IE2 binding. Mutation of otherbases in the third box reduces but does not abolish IE2 com-plex formation. In addition, bases within the last boxed regionof our consensus sequence (ATGTR) affect IE2 binding; how-ever, a specific sequence in this region is not required for IE2binding (37). If the IE2-binding site, like the EBV R-bindingsite, consists of two parts, it is interesting that gaps totaling 2bp were introduced into the L1.7 and BHV-4 homolog to theHSV-1 major DNA-binding protein gene (MDBP) IE2-bind-

ing fragment sequences to align them with the TK IE2-bindingfragment sequence. Therefore, if the region containing thegaps lies between two parts of the binding site, the two partsare separated by the same number of base pairs in the L1.7 andMDBP IE2-binding sites but are separated by an additional 2bp in the TK IE2-binding site. This observation is potentiallysignificant because the TK IE2-binding site binds IE2 lessefficiently than the L1.7 and MDBP IE2-binding sites (38).Therefore, less efficient binding of IE2 by the TK IE2-bindingsite might be due to suboptimal spacing of segments of thebinding site.

ACKNOWLEDGMENTS

This work was supported by grants from the Competitive ResearchGrants Office, USDA (90-32766-5783), and the Food Animal Healthand Disease Research Line Item, College of Veterinary Medicine,Auburn University.The excellent technical assistance of Karen Lamb, Patricia DeInno-

centes, and Chhavi Vig is gratefully acknowledged.

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FIG. 10. Alignment of sequences from IE2-binding fragments of threeBHV-4 promoters. The 31-bp IE2-binding fragment from the TK promoter waspreviously identified (38). The sequence from the BHV-4 homolog to the HSV-1MDBP promoter shown is part of a 53-bp IE2-binding fragment (33). Thisportion of the 53-bp fragment was chosen based on its similarity to the other twoIE2-binding fragments, but we have not precisely mapped the IE2-binding siteexperimentally. Alignment was done by inspection of sequences. Nucleotidesthat are the same in two or three sequences are indicated by shading and areshown in the consensus sequence. Nucleotides that are the same, or either allpurines or all pyrimidines, in all three IE2-binding fragments are boxed in theconsensus sequence. Abbreviations: R, purine; Y, pyrimidine.


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23. Manet, E., A. Rigolet, H. Gruffat, J.-F. Giot, and A. Sergeant. 1991. Domainsof the Epstein-Barr virus (EBV) transcription factor R required for dimer-ization, DNA binding and activation. Nucleic Acids Res. 19:2661–2667.

24. Mavromara-Nazos, P., S. Silver, J. Hubenthal-Voss, J. L. C. McKnight, andB. Roizman. 1986. Regulation of herpes simplex virus 1 genes: a genesequence requirements for transient induction of indicator genes regulatedby b or late g2 promoters. Virology 149:152–164.

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immediate-early transcript. J. Virol. 65:5211–5224.35. van Santen, V. L. 1993. Characterization of a bovine herpesvirus 4 immedi-

ate-early RNA encoding a homolog of the Epstein-Barr virus R transacti-vator. J. Virol. 67:773–784.

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characterization of an abundant, unique 1.7-kilobase bovine

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